The selective destruction of an individual cell or a specific cell type is often desirable in a variety of clinical settings. For example, it is a primary goal of cancer therapy to specifically destroy tumor cells, while leaving healthy cells and tissues intact and undamaged. A multitude of signal transduction pathways in the cell are linked to the cell's survival and/or death. Accordingly, the direct delivery of a pathway factor involved in cell survival or death can be used to contribute to the cell's maintenance or destruction. Similarly, specific factors may be delivered that stimulate immune effector cells in a tumor microenvironment, such as natural killer (NK) cells or cytotoxic T lymphocytes (CTLs), to attack and destroy tumor cells.
Cytokines are cell signaling molecules that participate in regulation of the immune system. When used in cancer therapy, cytokines can act as immunomodulatory agents that have anti-tumor effects and which can increase the immunogenicity of some types of tumors. However, rapid blood clearance and lack of tumor specificity require systemic administration of high doses of the cytokine in order to achieve a concentration of the cytokine at the tumor site sufficient to activate an immune response or have an anti-tumor effect. These high levels of systemic cytokine can lead to severe toxicity and adverse reactions.
For use in therapy, it is therefore desirable to specifically deliver a signal transduction pathway factor, such as a cytokine, to a specific site in vivo (e.g. a tumor or tumor microenvironment in the case of cancer therapy). This can be achieved by conjugating the factor to a targeting moiety, e.g. an antibody or an antibody fragment, specific for the site. Early strategies aimed at delivering signal transduction pathway factors, such as cytokines, to a specific site in vivo included immunoglobulin heavy chains conjugated to various cytokines, including lymphotoxin, tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), and granulocyte macrophage-colony stimulating factor (GM-CSF) (reviewed e.g. in Lode et al., Pharmacol Ther 80, 277-292 (1998)). Researchers observed that, not only were they able to target cytokines to specific sites in vivo, they were also able to take advantage of the fact that monoclonal antibodies have longer serum half-lives than most other proteins. Given the systemic toxicity associated with high doses of certain unconjugated cytokines, e.g. IL-2, the ability of an immunoglobulin-cytokine fusion protein to maximize therapeutically beneficial biological activities at a desired site, e.g. in a tumor, whilst keeping systemic side effects to a minimum at a lower dose led researchers to believe that immunoglobulin-cytokine immunoconjugates were optimal therapeutic agents.
Nevertheless, there are certain disadvantages associated with the immunoglobulin-cytokine immunoconjugtates known in the art. For example, these immunoconjugates have at least one cytokine coupled to each of the two immunoglobulin heavy chains, resulting in an immunoconjugate with bivalent target binding and two or more cytokine moieties (reviewed e.g. in Chang et al., Expert Opin Drug Discovery 4, 181-194 (2009), or Ortiz-Sanchez et al., Expert Opin Biol Ther 8, 609-632 (2008)). FIG. 1 depicts a conventional immunoglobulin-cytokine immunoconjugate as it is known in the art, where a cytokine is fused to the C-terminus of each of the two antibody heavy chains. Due to the presence of two or more cytokine moieties, such an immunoconjugate has a high avidity to the respective cytokine receptor (for example, picomolar affinity in the case of IL-2), and thus is targeted rather to the immune effector cells expressing the cytokine receptor than to the target antigen of the immunoglobulin (nM affinity) to which the cytokine is linked. Moreover, conventional immunoconjugates are known to be associated with infusion reactions (see e.g. King et al., J Clin Oncol 22, 4463-4473 (2004)), resulting at least partially from activation of cytokine receptors on immune effector cells in peripheral blood by the immunoconjugate's cytokine moieties.
Additionally, via their Fc domain, immunoglobulin-cytokine immunoconjugates can activate complement and interact with Fc receptors. This inherent immunoglobulin feature has been viewed unfavorably because therapeutic immunoconjugates may be targeted to cells expressing Fc receptors rather than the preferred antigen-bearing cells. Moreover, the simultaneous activation of cytokine receptors and Fc receptor signaling pathways leading to cytokine release, especially in combination with the long half-life of immunoglobulin fusion proteins, make their application in a therapeutic setting difficult due to systemic toxicity.
One approach to overcoming this problem is the use of immunoglobulin fragments devoid of an Fc domain, such as scFv or Fab fragments, in immunoconjugates. Examples of immunoglobulin fragment-cytokine immunoconjugates include the scFv-IL-2 immunoconjugate as set forth in PCT publication WO 2001/062298, the scFv-IL-12-scFv immunoconjugate as set forth in PCT publication WO 2006/119897 (wherein each of the two scFv fragments is connected to a subunit of the IL-12 heterodimer that is held together by disulfide bond(s)) or the Fab-IL-2-Fab immunoconjugates as set forth in PCT publication WO 2011/020783. Both the tumor-binding reactivity of the immunoglobulin parent molecule and the functional activity of the cytokine are maintained in most of these types of immunoconjugates, however the half-life of such constructs is considerably shorter than of immunoglobulin fusion proteins.
Therefore there remains a need for immunoconjugates with improved properties, for greater therapeutic effectiveness and a reduction in the number and severity of the side effects of these products (e.g., toxicity, destruction of non-tumor cells, etc.).
The present invention provides immunoglobulin-like immunoconjugates that exhibit improved efficacy, high specificity of action, reduced toxicity, and improved half-life and stability in blood relative to known immunoconjugates.